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Subcellular Distribution and In Situ Localization of the Acid Phosphatase of Entamoeba histolytica
Archives of Medical Research 31 (2000) S183–S184
Subcellular Distribution and In Situ Localization of the Acid Phosphatase of Entamoeba histolytica Javier Ventura-Juárez,* María Magdalena Aguirre-García** and Patricia Talamás-Rohana** *Departamento de Morfología, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Aguascalientes, Mexico **Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados del I.P.N. (Cinvestav), Mexico City, Mexico
Key Words: Entamoeba histolytica, Acid phosphatase, Immunolocalization, Pathogenicity.
Introduction In invasive amebiasis, attempts have been made to correlate the biochemical and functional properties observed in vitro with the virulence of trophozoites, using as a measure of virulence their ability to produce amebic hepatic abscess in animal models (1). Invasion of host tissues by the trophozoites is accompanied by contact-dependent cell lysis and phagocytosis, as well as by the secretion of lytic components (2). Recently, we reported that Entamoeba histolytica contains and secretes an acid phosphatase (AP), in contrast with Entamoeba dispar, which possesses the activity but is unable to secrete it (3). Additionally, we have described the purification and properties of the E. histolytica acid phosphatase (4). In this work, we present data concerning the subcellular localization by immunofluorescence of the AP in fixed trophozoites, as well as its in situ localization in infected tissue by using polyclonal antibodies prepared against purified AP. Materials and Methods Cell culture. E. histolytica HM-1:IMSS strain was grown as described (4). Purification of AP. The enzyme was purified as reported previously (4).
Immunofluorescence analysis. Trophozoites were fixed with 4% paraformaldehyde for 1 h at 37⬚C and permeabilized with PBS-T (0.2% Triton X-100) for 20 min at room temperature. Cells were incubated with anti-AP antibodies (1:200), then incubated with a FITC-conjugated secondary goat-antimouse antibody (1:50), and visualized by confocal microscopy. Immunohistochemistry for light and electron microscopy detection of AP. For abscess development, the reported technique was followed (5). After different times postinoculation, animals were anesthetized with sodium pentobarbital (94.5 mg/kg of body weight) and the portal vein uncovered to perfuse the liver with 4% paraformaldehyde, 1% glutaraldehyde, and 15% picric acid in 0.1 M sodium cacodylate buffer for 30 min. The liver was dissected and small fragments 0.1–0.2 cm thick were transferred to the same fixative and incubated for at least 2 h. For light microscopy, paraffin sections were treated as reported previously (5), using the anti-AP antibody at a 1:100 dilution. For electron microscopy, liver samples were included in LR-White, the anti-AP antibody used at a 1:50 dilution, and the secondary antibody (a 20-nm coloidal gold-labeled goat-antimouse IgG) at a 1:20 dilution (5).
Results and Discussion Polyclonal anti-AP antibody production. Balb/c mice were immunized subcutaneously at least four times with DEAEenriched fractions. Samples were emulsified with Titer Max the first and second times, and with Melox at subsequent times. Address reprint requests to: Patricia Talamás-Rohana, Departamento de Patología Experimental, Cinvestav, Av. Instituto Politécnico Nacional #2508, Col. San Pedro Zacatenco, 07360 México, D.F., México. Tel.: (⫹525) 747-3800, ext. 5635; FAX: (⫹525) 747-9890; E-mail: ptr@gwpat. pat.cinvestav.mx Presenting author: Javier Ventura-Juárez.
Subcellular localization of E. histolytica AP. To learn more about the subcellular localization of AP in trophozoites, we prepared polyclonal monospecific antibodies against AP. DEAE-eluted material was used directly to immunize mice. We found that the antibodies recognized a high-molecularweight component of approximately 120 kDa present in solubilized fraction (data not shown). Immunofluorescence localization of E. histolytica AP. The staining pattern of the anti-AP antibody showed a peripheral staining in nonpermeabilized cells, whereas in permeabi-
0188-4409/00 $–see front matter. Copyright © 2000 IMSS. Published by Elsevier Science Inc. PII S0188-4409(00)00 2 0 8 - 3
S184
Ventura-Juárez et al./ Archives of Medical Research 31 (2000) S183–S184
lized trophozoites, the antibody stained the vacuoles membranes and also some cytoplasmic granular components (Figure 1a and b, arrows). This staining pattern coincides with the distribution of AP by subcellular fractionation (3). Immunodetection of E. histolytica AP in infected tissue. When the anti-AP antibody was used to detect AP in infected tissue, as early as 5 min postinoculation, all the trophozoites seen in a hepatic sinusoid were intensively labeled, as well as the endothelial cell layer (Figure 1c). This is opposed to some data (not shown) obtained with an AP chromogenic substrate that displayed a heterogeneous dis-
tribution of the enzymatic activity among cell population in axenic culture. As shown by electron microscopy (Figure 1d–g), the labeled antibody is localized in the vacuolar system of the trophozoites, in the interface ameba–host cell, and in engulfed material inside polymorphonuclear cells. Vacuoles show their inner border decorated by the antibody (Figure 1e and f); sometimes, the label is found decorating small granules that seem to be going through a secretion process, as shown in Figure 1e (double arrow). Figure 1g shows the interface between a trophozoite and a polymorphonuclear cell full of labeled material. Figure 1d corresponds to a negative control using only the secondary antibody. The staining of normal noninfected tissue with the anti-AP antibody did not show crossreactivity (data not shown), confirming that the antibody is recognizing amebic components present in both the trophozoite and in the parasite–host cell interface. Despite the differences between SAP and MAP, the fact that the anti-MAP antibody recognized both enzymes in situ may suggest that both activities correspond to one entity that can present structural (molecular weight) and functional (optimum pH and behavior with chemical compounds) modifications. Another possibility is that these enzymes are different but share some antigenic epitopes. These results support the hypothesis that amebic AP could be an important in vivo element in the pathogenic mechanism. Acknowledgments We thank M. Anaya for the preparation of anti-AP antibody. This work was partially supported by grant 3687-M9607 from Conacyt (Mexico) and grant 98-128RG/BIO/LA from the Third World Academy of Sciences. M.M.A.G. was a recipient of a fellowship from Conacyt (Mexico).
References
Figure 1. Subcellular localization of E. histolytica MAP. Indirect immunofluorescence localization by confocal microscopy of E. histolytica AP on nonpermeabilized (a) and permeabilized (b) trophozoites. In situ localization of AP in E. histolytica-infected hamster after 5 min postinoculation (c). External membranes and vacuoles (arrows) of trophozoites as well as the sinusoidal endothelial cells (double arrows) show a positive reaction to the presence of AP. In situ localization of AP in E. histolytica-infected hamster by electron microscopy. Negative control incubated only with the secondary gold-labeled goat-antimouse IgG (d). Positive reaction for AP (e–f). (H), hepatocytes; (P), polymorphonuclear cell; (T), trophozoites; (V), vacuoles; (arrow), labeled vacuoles; (arrowheads), labeled granules; (double arrow and *), interface between a trophozoite and a host cell.
1. Tsutsumi V, Ramírez-Rosales A, Lanz-Mendoza H, Shibayama M, Chávez B, Rangel-López E, Martínez-Palomo A. Entamoeba histolytica: erythrophagocytosis, collagenolysis, and liver abscess production as virulence markers. Trans R Soc Trop Med Hyg 1992;86:170. 2. Martínez-Palomo A, Kretschmer R, Meza Gómez Palacio I. Entamoeba histolytica and amebiasis. In: Warren K, editor. Immunology and molecular biology of parasitic infections. Boston, MA, USA: Blackwell Scientific Publications;1993. p. 143. 3. Talamás-Rohana P, Aguirre-García MM, Anaya-Ruíz M, RosalesEncina JL. Entamoeba dispar contains but does not secrete acid phosphatase as does Entamoeba histolytica. Exp Parasitol 1999;92:219. 4. Aguirre-García MM, Cerbón J, Talamás-Rohana P. Purification and properties of an acid phosphatase from Entamoeba histolytica HM1:IMSS. Int J Parasitol 2000 (In press). 5. Ventura-Juárez J, Campos-Rodríguez R, Rodríguez-Martínez HA, Rodríguez-Reyes A, Martínez-Palomo A, Tsutsumi V. Human amebic liver abscess: expression of intracellular adhesion molecules and of von Willebrand factor in endothelial cells. Parasitol Res 1997;83:510.
Subcellular Distribution and In Situ Localization of the Acid Phosphatase of Entamoeba histolytica Javier Ventura-Juárez,* María Magdalena Aguirre-García** and Patricia Talamás-Rohana** *Departamento de Morfología, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Aguascalientes, Mexico **Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados del I.P.N. (Cinvestav), Mexico City, Mexico
Key Words: Entamoeba histolytica, Acid phosphatase, Immunolocalization, Pathogenicity.
Introduction In invasive amebiasis, attempts have been made to correlate the biochemical and functional properties observed in vitro with the virulence of trophozoites, using as a measure of virulence their ability to produce amebic hepatic abscess in animal models (1). Invasion of host tissues by the trophozoites is accompanied by contact-dependent cell lysis and phagocytosis, as well as by the secretion of lytic components (2). Recently, we reported that Entamoeba histolytica contains and secretes an acid phosphatase (AP), in contrast with Entamoeba dispar, which possesses the activity but is unable to secrete it (3). Additionally, we have described the purification and properties of the E. histolytica acid phosphatase (4). In this work, we present data concerning the subcellular localization by immunofluorescence of the AP in fixed trophozoites, as well as its in situ localization in infected tissue by using polyclonal antibodies prepared against purified AP. Materials and Methods Cell culture. E. histolytica HM-1:IMSS strain was grown as described (4). Purification of AP. The enzyme was purified as reported previously (4).
Immunofluorescence analysis. Trophozoites were fixed with 4% paraformaldehyde for 1 h at 37⬚C and permeabilized with PBS-T (0.2% Triton X-100) for 20 min at room temperature. Cells were incubated with anti-AP antibodies (1:200), then incubated with a FITC-conjugated secondary goat-antimouse antibody (1:50), and visualized by confocal microscopy. Immunohistochemistry for light and electron microscopy detection of AP. For abscess development, the reported technique was followed (5). After different times postinoculation, animals were anesthetized with sodium pentobarbital (94.5 mg/kg of body weight) and the portal vein uncovered to perfuse the liver with 4% paraformaldehyde, 1% glutaraldehyde, and 15% picric acid in 0.1 M sodium cacodylate buffer for 30 min. The liver was dissected and small fragments 0.1–0.2 cm thick were transferred to the same fixative and incubated for at least 2 h. For light microscopy, paraffin sections were treated as reported previously (5), using the anti-AP antibody at a 1:100 dilution. For electron microscopy, liver samples were included in LR-White, the anti-AP antibody used at a 1:50 dilution, and the secondary antibody (a 20-nm coloidal gold-labeled goat-antimouse IgG) at a 1:20 dilution (5).
Results and Discussion Polyclonal anti-AP antibody production. Balb/c mice were immunized subcutaneously at least four times with DEAEenriched fractions. Samples were emulsified with Titer Max the first and second times, and with Melox at subsequent times. Address reprint requests to: Patricia Talamás-Rohana, Departamento de Patología Experimental, Cinvestav, Av. Instituto Politécnico Nacional #2508, Col. San Pedro Zacatenco, 07360 México, D.F., México. Tel.: (⫹525) 747-3800, ext. 5635; FAX: (⫹525) 747-9890; E-mail: ptr@gwpat. pat.cinvestav.mx Presenting author: Javier Ventura-Juárez.
Subcellular localization of E. histolytica AP. To learn more about the subcellular localization of AP in trophozoites, we prepared polyclonal monospecific antibodies against AP. DEAE-eluted material was used directly to immunize mice. We found that the antibodies recognized a high-molecularweight component of approximately 120 kDa present in solubilized fraction (data not shown). Immunofluorescence localization of E. histolytica AP. The staining pattern of the anti-AP antibody showed a peripheral staining in nonpermeabilized cells, whereas in permeabi-
0188-4409/00 $–see front matter. Copyright © 2000 IMSS. Published by Elsevier Science Inc. PII S0188-4409(00)00 2 0 8 - 3
S184
Ventura-Juárez et al./ Archives of Medical Research 31 (2000) S183–S184
lized trophozoites, the antibody stained the vacuoles membranes and also some cytoplasmic granular components (Figure 1a and b, arrows). This staining pattern coincides with the distribution of AP by subcellular fractionation (3). Immunodetection of E. histolytica AP in infected tissue. When the anti-AP antibody was used to detect AP in infected tissue, as early as 5 min postinoculation, all the trophozoites seen in a hepatic sinusoid were intensively labeled, as well as the endothelial cell layer (Figure 1c). This is opposed to some data (not shown) obtained with an AP chromogenic substrate that displayed a heterogeneous dis-
tribution of the enzymatic activity among cell population in axenic culture. As shown by electron microscopy (Figure 1d–g), the labeled antibody is localized in the vacuolar system of the trophozoites, in the interface ameba–host cell, and in engulfed material inside polymorphonuclear cells. Vacuoles show their inner border decorated by the antibody (Figure 1e and f); sometimes, the label is found decorating small granules that seem to be going through a secretion process, as shown in Figure 1e (double arrow). Figure 1g shows the interface between a trophozoite and a polymorphonuclear cell full of labeled material. Figure 1d corresponds to a negative control using only the secondary antibody. The staining of normal noninfected tissue with the anti-AP antibody did not show crossreactivity (data not shown), confirming that the antibody is recognizing amebic components present in both the trophozoite and in the parasite–host cell interface. Despite the differences between SAP and MAP, the fact that the anti-MAP antibody recognized both enzymes in situ may suggest that both activities correspond to one entity that can present structural (molecular weight) and functional (optimum pH and behavior with chemical compounds) modifications. Another possibility is that these enzymes are different but share some antigenic epitopes. These results support the hypothesis that amebic AP could be an important in vivo element in the pathogenic mechanism. Acknowledgments We thank M. Anaya for the preparation of anti-AP antibody. This work was partially supported by grant 3687-M9607 from Conacyt (Mexico) and grant 98-128RG/BIO/LA from the Third World Academy of Sciences. M.M.A.G. was a recipient of a fellowship from Conacyt (Mexico).
References
Figure 1. Subcellular localization of E. histolytica MAP. Indirect immunofluorescence localization by confocal microscopy of E. histolytica AP on nonpermeabilized (a) and permeabilized (b) trophozoites. In situ localization of AP in E. histolytica-infected hamster after 5 min postinoculation (c). External membranes and vacuoles (arrows) of trophozoites as well as the sinusoidal endothelial cells (double arrows) show a positive reaction to the presence of AP. In situ localization of AP in E. histolytica-infected hamster by electron microscopy. Negative control incubated only with the secondary gold-labeled goat-antimouse IgG (d). Positive reaction for AP (e–f). (H), hepatocytes; (P), polymorphonuclear cell; (T), trophozoites; (V), vacuoles; (arrow), labeled vacuoles; (arrowheads), labeled granules; (double arrow and *), interface between a trophozoite and a host cell.
1. Tsutsumi V, Ramírez-Rosales A, Lanz-Mendoza H, Shibayama M, Chávez B, Rangel-López E, Martínez-Palomo A. Entamoeba histolytica: erythrophagocytosis, collagenolysis, and liver abscess production as virulence markers. Trans R Soc Trop Med Hyg 1992;86:170. 2. Martínez-Palomo A, Kretschmer R, Meza Gómez Palacio I. Entamoeba histolytica and amebiasis. In: Warren K, editor. Immunology and molecular biology of parasitic infections. Boston, MA, USA: Blackwell Scientific Publications;1993. p. 143. 3. Talamás-Rohana P, Aguirre-García MM, Anaya-Ruíz M, RosalesEncina JL. Entamoeba dispar contains but does not secrete acid phosphatase as does Entamoeba histolytica. Exp Parasitol 1999;92:219. 4. Aguirre-García MM, Cerbón J, Talamás-Rohana P. Purification and properties of an acid phosphatase from Entamoeba histolytica HM1:IMSS. Int J Parasitol 2000 (In press). 5. Ventura-Juárez J, Campos-Rodríguez R, Rodríguez-Martínez HA, Rodríguez-Reyes A, Martínez-Palomo A, Tsutsumi V. Human amebic liver abscess: expression of intracellular adhesion molecules and of von Willebrand factor in endothelial cells. Parasitol Res 1997;83:510.